An In Vitro Comparison of PMMA and Calcium Sulfate as Carriers for the Local Delivery of Gallium(III) Nitrate to Staphylococcal Infected Surgical Sites

Abstract

Antibiotic-loaded bone cements, including poly(methyl methacrylate) (PMMA) and calcium sulfate (CaSO4), are often used for treatment of orthopaedic infections involving Staphylococcus spp., although the effectiveness of this treatment modality may be limited due to the emergence of antimicrobial resistance and/or the development of biofilms within surgical sites. Gallium(III) is an iron analog capable of inhibiting essential iron-dependent pathways, exerting broad antimicrobial activity against multiple microorganisms, including Staphylococcus spp. Herein, we evaluated PMMA and CaSO4 as carriers for delivery of gallium(III) nitrate (Ga(NO3)3) to infected surgical sites by assessing the release kinetics subsequent to incorporation and antimicrobial activity against S. aureus and S. epidermidis. PMMA and to a lesser extent CaSO4 were observed to be compatible as carriers for Ga(NO3)3, eluting concentrations with antimicrobial activity against planktonic bacteria, inhibiting bacterial growth, and preventing bacterial colonization of beads, and effective against established bacterial biofilms of S. aureus and S. epidermidis. Collectively, our in vitro results indicate that PMMA is a more suitable carrier compared to CaSO4 for delivery of Ga(NO3)3; moreover they provide evidence for the potential use of Ga(NO3)3 with PMMA as a strategy for the prevention and/or treatment for orthopaedic infections.

Figure 1

In vitro antimicrobial activities of Ga(NO₃)₃. Activity of Ga(NO₃)₃, 0.5–512 μM, against planktonic bacteria (a) and biofilms (b) of Staphylococcus epidermidis ATCC 12228 and Staphylococcus aureus ATCC 29213 following overnight exposure to increasing concentrations, in MHB II (0.3 g/L) broth in 96-well plates. Data is representative of mean ± SD of three independent experiments. Statistical analysis was performed using Student's t-test; ∗ indicates p < 0.05 relative to the untreated control group; Student's t-test.

Figure 2

Characterization of Ga(NO₃)₃ release kinetics from PMMA and CaSO₄ beads. Absolute release of Ga(NO₃)₃ from PMMA (a) and CaSO₄ (b) beads, loaded at 2.4, 4.7, 9.09, and 13% (w/w), over 7 days performed in PBS is shown. The percent cumulative release of the total amount of Ga(NO₃)₃ (by weight) loaded into PMMA (c) and CaSO₄ (d) beads is shown. Values are reported as the mean ± SD of n = 3 samples.

Figure 3

Activity of Ga(NO₃)₃ loaded PMMA and CaSO₄ beads on planktonic bacteria. Antimicrobial activity of PMMA (a-b) and CaSO₄ (c-d) loaded Ga(NO₃)₃ beads on bacterial cultures of S. aureus ATCC 12913 and S. epidermidis ATCC 12228 over time. Data are expressed as log reduction relative to empty (0.0%) PMMA/CaSO₄ beads. Values are reported as the mean ± SD. Statistical analysis was performed using an analysis of variance (ANOVA) with Dunnett's post hoc test; ∗ indicates p < 0.05 relative to control.

Figure 4

Effect of Ga(NO₃)₃ incorporation on bacterial colonization of PMMA and CaSO₄. Colonization of Ga(NO₃)₃ loaded PMMA (a-b) or CaSO₄ (c-d), 2.4%, 4.7%, 9.09%, and 13.0% (w/w), by S. aureus ATCC 12913 and S. epidermidis ATCC 12228 over time, as determined by CFU counts (log₁₀ CFU/mL per bead). Data is representative of mean ± SD. Statistical analysis was performed using an analysis of variance (ANOVA) with Dunnett's post hoc test; ∗ indicates p < 0.05 relative to control.

Figure 5

Activity of Ga(NO₃)₃ loaded PMMA beads on established biofilms. Effects of Ga(NO₃)₃ loaded (a) PMMA and (b) CaSO₄ beads (% w/w) on established biofilms of S. aureus ATCC 12913 and S. epidermidis ATCC 12228 following overnight exposure. Data is expressed as the log reductions (mean ± SD) relative to biofilms treated with unloaded control beads. Statistical analysis was performed using an analysis of variance (ANOVA) with Dunnett's post hoc test; ∗ indicates p < 0.05 relative to control.